RESEARCH ARTICLE Open Access

Evaluation of the Etest and disk diffusionmethod for detection of the activity ofceftazidime-avibactam againstEnterobacterales and Pseudomonasaeruginosa in ChinaQi Wang, Feifei Zhang, Zhanwei Wang, Hongbin Chen, Xiaojuan Wang, Yawei Zhang, Shuguang Li andHui Wang*

Abstract

Background: Ceftazidime-avibactam was approved in China in 2019 for treating complicated intra-abdominalinfections, hospital-acquired pneumonia, ventilator-associated pneumonia, and infections caused by Enterobacteralesand Pseudomonas aeruginosa for which treatment options are limited. However, no currently available commercialsystems have been approved for antimicrobial susceptibility testing of ceftazidime-avibactam in China. Here, weevaluated the Etest and disk diffusion method for detecting the activity of ceftazidime-avibactam againstEnterobacterales and P. aeruginosa in China.

Results: In total, 194 Enterobacterales and 77 P. aeruginosa isolates, which were divided into a random selectiongroup (140 Enterobacterales and 46 P. aeruginosa isolates) and stock group (54 Enterobacterales and 31 P. aeruginosaisolates), were assessed by the Etest, disk diffusion and broth microdilution methods. Minimum inhibitoryconcentrations and zone diameters were interpreted according to the CLSI supplement M100 30th edition. For all271 tested isolates, no very major errors were found by using Etest, whereas the overall major error rate was 2.0%(4/203). The overall categorical agreement rates of Etest for Enterobacterales and P. aeruginosa were 99.5% (193/194)and 96.1% (74/77), respectively, and the essential agreement rates were 95.9% (186/194) and 94.8% (73/77),respectively. The disk diffusion method showed that the very major error and major error rates were 1.5% (3/204)and 2.5% (5/203), respectively. Overall categorical agreement rates values of the disk diffusion method forEnterobacterales and P. aeruginosa were 98.5% (191/194) and 93.5% (72/77) compared with broth microdilution,respectively.

Conclusions: For Enterobacterales and P. aeruginosa, both the Etest and disk diffusion method showed acceptableperformance as alternatives to the standard broth microdilution method for clinical treatment interpretation.Application of the disk diffusion method in Enterobacterales was slightly better than that in P. aeruginosa.

Keywords: Ceftazidime-avibactam, Etest, Broth microdilution, Disk diffusion

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* Correspondence: whuibj@163.comDepartment of Clinical Laboratory, Peking University People’s Hospital,Beijing 100044, China

Wang et al. BMC Microbiology (2020) 20:187 https://doi.org/10.1186/s12866-020-01870-z

BackgroundGram-negative bacilli, particularly carbapenem-resistantEnterobacterales (CRE) and Pseudomonas aeruginosa,exhibit major antimicrobial resistance worldwide, includ-ing in European countries, the United States (USA), andChina [1–3]. The approval of ceftazidime-avibactam forclinical use in Europe and the USA has provided newtreatment options for CRE-infected patients, particularlythose with serine-carbapenemase resistance mechanisms[4, 5]. Ceftazidime-avibactam was approved in China in2019 for treating complicated intra-abdominal infec-tions, hospital-acquired pneumonia, ventilator-associatedpneumonia, and infections caused by Enterobacteralesand P. aeruginosa without enough effective treatmentoptions. In China, there is currently no automated sys-tem for antimicrobial susceptibility testing ofceftazidime-avibactam. Ceftazidime-avibactam suscepti-bility tests are urgently needed to facilitate appropriatetargeted treatment in patients with infections caused bymultidrug-resistant Enterobacterales and P. aeruginosa.Although ceftazidime-avibactam has excellent in vitroactivity against carbapenem-resistant Klebsiella pneumo-niae (CRKP) and P. aeruginosa isolates, a few resistantisolates remain during treatment or nosocomial infectiontransmission [6–8]. Therefore, the susceptibility test re-sults for ceftazidime-avibactam are even more critical.In most laboratories in China, performing the standard

broth microdilution (BMD) method is challenging.Therefore, alternative methods are needed to reliably de-termine ceftazidime-avibactam susceptibility. In thisstudy, we evaluated two antimicrobial susceptibility test-ing methods for ceftazidime-avibactam, i.e., the Etestand disk diffusion method with the reference BMD, toevaluate whether these simple methods can be utilizedfor testing ceftazidime-avibactam in the clinical setting.

ResultsEtest versus the BMDAccording to the BMD method, 24.2% (47/194) of Enter-obacterales and 28.4% (21/74) of P. aeruginosa were re-sistant to ceftazidime-avibactam. For all 194Enterobacterales isolates, no very major error (VME)were found using the Etest. One isolate in the stockgroup was classified as resistant by Etest but susceptibleby BMD. The major error (ME) rate was 0.7% (1/147) inEnterobacterales. As shown in Table 1, the overall cat-egorical agreement (CA) rate was 99.5%, and the overallessential agreement (EA) rate was 95.9%. The CA rate ofthe stock group was 98.1%, and that in the random se-lection group was 97.9%. When comparing Etest resultswith the BMD results, the MICs of nine isolatesexceeded the two-fold dilution. As shown in Fig. 1, theEtest minimum inhibitory concentrations (MICs) of 102(52.5%) isolates were consistent with the BMD MICs.The Etest MICs of 71 (36.6%) isolates were one-fold di-lution higher than the BMD MICs. Only 12 (6.2%) iso-lates showed Etest MICs that were one-fold dilutionlower than the BMD MICs.For 77 P. aeruginosa isolates, no VME were found

using the Etest method. Three isolates in the stockgroup were classified as resistant by Etest but susceptibleby BMD. As shown in Table 1, the overall CA and EArates were 96.1% and 94.8%, respectively. The CA ratesin the stock and random selection groups were 90.3%and 100%, respectively. As shown in Fig. 2, the EtestMICs of 45 (58.4%) isolates were consistent with thoseobtained by the BMD. The Etest MICs of 22 (28.6%) iso-lates were one-fold dilution higher than those obtainedby the BMD, whereas those of six (7.8%) isolates wereone-fold dilution lower than the MICs obtained by theBMD. For one strain, the Etest MIC was two-fold

Table 1 Evaluation of essential and categorical agreement between the BMD method and Etest or disk diffusion method foranalysis of ceftazidime-avibactam antimicrobial susceptibility

Organism No. ofisolatestested

No. ofresistantisolatesby BMD

E-test Disk diffusion

No. (%) ofCA

No. (%) ofEA

No. (%) ofVME

No. (%) ofME

No. (%) ofCA

No. (%) ofVME

No. (%) ofME

Enterobacterales

Random selection group 140 7 140 (100) 137 (97.9) 0 (0) 0 (0) 140 (100) 0 (0) 0 (0)

Stock group 54 40 53 (98.1) 49 (90.7) 0 (0) 1 (7.1) 51 (94.4) 2 (5.0) 1 (7.1)

Total in Enterobacterales 194 47 193 (99.5) 186 (95.9) 0 (0) 1 (0.7) 191 (98.5) 2 (4.3) 1 (0.7)

P. aeruginosa

Random selection group 46 6 46 (100) 45 (97.8) 0 (0) 0 (0) 43 (93.5) 0 (0) 3 (7.5)

Stock group 31 15 28 (90.3) 28 (90.3) 0 (0) 3 (18.7) 29 (93.5) 1 (6.7) 1 (6.3)

Total in P. aeruginosa 77 21 74 (96.1) 73 (94.8) 0 (0) 3 (5.4) 72 (93.5) 1 (4.8) 4 (7.1)

Total in all testedisolates

271 68 267 (98.5) 259 (95.6) 0 (0) 4 (2.0) 263 (97.0) 3 (1.5) 5 (2.5)

EA essential agreement, CA categorical agreement, VME very major error (false susceptible), ME major error (false resistant)

Wang et al. BMC Microbiology (2020) 20:187 Page 2 of 7

dilutions higher than that obtained by the BMD. ThreeMEs appeared in the stock group when the Etest methodwas used.

Disk diffusion method versus BMDA comparison of the disk diffusion method and BMDresults for 194 Enterobacterales isolates are shown inFig. 3. The overall CA rate in the 194 Enterobacteralesisolates was 98.5%. Two VMEs and 1 ME were foundusing the disk diffusion method in the stock group; allwere carbapenem-resistant K. pneumoniae carbapene-mase (KPC)-producing isolates. No VME or ME werefound using the disk diffusion method in the random se-lection group. There were 22 isolates of Enterobacteraleswith zone diameters between 19 and 22mm. Forty-oneof the 47 resistant isolates obtained by BMD showedzone diameters in the range of 13–20mm.Comparison of the disk diffusion method and BMD

for 77 P. aeruginosa isolates showed that the ME% was7.1%. As shown in Table 1, the overall CA rate was93.5%. Moreover, as shown in Fig. 4, when the zonediameter was 20 mm, the MICs obtained by the BMDmethod were 4, 8, 16, 32, and 64 μg/mL.

DiscussionIn the past decade, the incidence of CRE, particularlyCRKP, has increased significantly in China. The latestChina Antimicrobial Resistance Surveillance System datashowed that the incidence of CRKP nationwide was ashigh as 10.1% (http://www.carss.cn/Report/Details?aId=648). However, few active antibacterial agents, such astigecycline and colistin, are available to treat CRKP inthe clinical setting, resulting in high mortality worldwide[9, 10]. Previous molecular epidemiological data revealedthat more than 70% of CRE isolated in China from 2012to 2016 produce KPC-type carbapenemases [11].Ceftazidime-avibactam, a drug with potent antibacterialactivity against serine-carbapenemase, was approved foruse in China in 2019. Recent retrospective studiesshowed that ceftazidime-avibactam treatment of CRKPand P. aeruginosa infection in patients who have under-gone solid organ transplantation improves clinicalsuccess rates [12, 13]. Despite these promising findings,ceftazidime-avibactam antimicrobial susceptibility testresults are essential for the clinical use of this treatment.Additionally, commercial automated systems in theclinical microbiology laboratory cannot be used for

Fig. 1 Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Enterobacterales. Dotted lines represent the susceptibilitybreakpoint for ceftazidime-avibactam. VME: very major error (false susceptible); ME: major error (false resistant). The gray background indicatesthat the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicates that amajor error occurred in the MIC of the Etest compared with the MIC of the BMD

Wang et al. BMC Microbiology (2020) 20:187 Page 3 of 7

antimicrobial susceptibility testing of ceftazidime-avibactam in China.This is the first study to compare the Etest, disk diffu-

sion method, and BMD to detect ceftazidime-avibactamsusceptibility in China. We use BMD as a standard

clinical method for comparison with the other methods.Compared with the standard BMD method, no VMEwere found using the Etest method. The results of EtestMICs and BMD MICs were reasonably well correlatedfor both Enterobacterales and P. aeruginosa. The overall

Fig. 3 Scatter plot of ceftazidime-avibactam zone diameters versus BMD MICs against Enterobacterales. Dotted lines represent the susceptibilitybreakpoint for ceftazidime-avibactam. VME: very major error (false susceptible); ME: major error (false resistant). The yellow background indicatesthat a major error occurred for the disk diffusion method compared with the BMD. The red background indicates that two very major errorsoccurred when the disk diffusion method was compared with the BMD

Fig. 2 Scatter plot of ceftazidime-avibactam Etest MICs versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent thesusceptibility breakpoint for ceftazidime-avibactam. VME: very major error (false susceptible); ME: major error (false resistant). The gray backgroundindicates that the MIC of the Etest did not satisfy the essential agreement compared with the MIC of the BMD; the yellow background indicatesthat three major errors occurred in the MIC of the Etest compared with the MIC of the BMD

Wang et al. BMC Microbiology (2020) 20:187 Page 4 of 7

EA% of 271 tested isolates was 95.6%. Compared withthe BMD method, the Etest method exhibited an excel-lent linear correlation, supporting the use of this ap-proach as an alternative to the standard clinical methodwithout considering economic costs. The Etest resultswere similar to those of previous research findings [14].A study of the ceftazidime-avibactam Etest in Germanyreported excellent results in terms of EA and CA forsusceptibility testing of Enterobacterales and P. aerugi-nosa [15]. A total of 140 Enterobacterales and 60 P. aer-uginosa were examined; the total EA% and CA% were99.0% and 99.5%, respectively.The disk diffusion method is easy to implement in the

clinical setting from an economic perspective. The CA%values of the ceftazidime-avibactam disk diffusionmethod against Enterobacterales and P. aeruginosa were98.5% and 93.5%, respectively. However, a previous studyby Shields et al. showed that 28% of ceftazidime-avibactam susceptible CRE isolates were classified as re-sistant by disk diffusion. The ME% is significantly higherthan that obtained in our study [14]. This difference indata may be related to differences in the disk manufac-turer. Indeed, our results showed that application of thedisk diffusion method was more appropriate for Entero-bacterales than for P. aeruginosa. Notably, however, thedisk diffusion method did not exhibit an excellent linearcorrelation with BMD. Moreover, for both Enterobacter-ales or P. aeruginosa against ceftazidime-avibactam, thedisk diffusion method tends to show falsely susceptibleresults. As the sample size in our study was small, thenumber of VME was low. Therefore, clinical verificationof larger sample sizes is needed.

Based on our results, when the zone diameters ofceftazidime-avibactam against Enterobacterales and P.aeruginosa were 20–21mm, BMD testing should beperformed to avoid false-susceptible or false-resistant re-sults. Our results were consistent with those of otherstudies [16, 17]. This should also be considered whenusers refer to the new version of CLSI M100, which sug-gests using the disk diffusion method for Enterobacter-ales [18]. The CLSI recommends that MICs should bedetermined if the zone for Enterobacterales isolates is20–22mm. CLSI does not have a similar comment forP. aeruginosa.

ConclusionsIn conclusion, for Enterobacterales and P. aeruginosa,ceftazidime-avibactam Etest and the disk diffusionmethod showed acceptable performance as alternativesto the BMD method for clinical treatment interpretation.Application of the disk diffusion method for Enterobac-terales was slightly better than that for P. aeruginosa.

MethodsBacterial groupsIsolates were divided into two groups, i.e., random selec-tion group and stock group (Table S1). For the randomselection group, we randomly selected 140 Enterobacter-ales and 46 P. aeruginosa isolates from clinical non-repeated isolates obtained from Peking UniversityPeople’s Hospital. Among these isolates, 59.3% (83/140)of Enterobacterales and 56.5% (26/46) of P. aeruginosaisolates were defined as fresh clinical isolates obtainedwithin 1 month prior to testing (November 2019 to

Fig. 4 Scatter plot of ceftazidime-avibactam zone diameters versus BMD MICs against Pseudomonas aeruginosa. Dotted lines represent thesusceptibility breakpoint for ceftazidime-avibactam. VME: very major error (false susceptible); ME: major error (false resistant). The yellowbackground indicates that four major errors occurred for the disk diffusion method compared with the BMD. The red background indicates that avery major error occurred when the disk diffusion method was compared with the BMD

Wang et al. BMC Microbiology (2020) 20:187 Page 5 of 7

March 2020). Based on the collection of fresh clinicalisolates, we conducted three batches of antimicrobialsusceptibility tests, each in parallel with three methods(BMD, Etest, and disk diffusion method). The first testwas conducted in December 2019, and the testing iso-lates were isolated within 1 month before the test date.The second and third tests were conducted in January2020 and March 2020, respectively, and the test isolateswere isolated within 1 month before the test date. Theremaining isolates in the random selection group wereobtained from the strain repository of Peking UniversityPeople’s Hospital from January 2018 to October 2019.The 140 isolates of Enterobacterales used for testing in-cluded 13 species, i.e., 25 K. pneumoniae, 19 Escherichiacoli, 18 Proteus mirabilis, 17 Enterobacter cloacae, 16Serratia marcescens, 15 Citrobacter freundii, 14 K. oxy-toca, 4 Proteus vulgaris, 3 Morganella morganii, 3 Provi-dencia stuartii, 2 Providencia rettgeri, 2 K. aerogenes, and2 Citrobacter koseri isolates. For the stock group, we se-lected 54 isolates of Enterobacterales from 15 hospitalsin the CRE China-Network from January 2015 to Octo-ber 2019 and requested MICs of ceftazidime-avibactamof 2–>256 μg/mL. Among these isolates, six (11.1%)showed MICs for ceftazidime-avibactam of 8–16 μg/mL,and 15 isolates (27.8%) showed MICs of 4–32 μg/mL.These isolates with known MICs were mainly used toverify the accuracy of the value near the ceftazidime-avibactam breakpoint. The carbapenem-resistance genespresent in these isolates were determined in previousstudies [11]. The 54 isolates of Enterobacterales used inthis study included 29 K. pneumoniae (18 with blaKPCand 10 with blaNDM), 12 E. coli (2 with blaKPC and 7with blaNDM), 8 E. cloacae (1 with blaKPC, 5 withblaNDM, 1 with blaIMP, and 1 with blaVIM), 3 K. oxytoca(2 with blaIMP and 1 with blaNDM), and 2 Citrobacterfreundii (1 with blaIMP and 1 with blaNDM).We selected 31 isolates of P. aeruginosa from the 8

hospitals involved in the Chinese Antimicrobial Resist-ance Surveillance of Nosocomial infections 2018 projectas stock group isolates. The MICs of ceftazidime-avibactam were 2–>256 μg/mL. Among these isolates, 12(38.7%) had MICs for ceftazidime-avibactam between 8and 16 μg/mL and 25 (80.6%) had MICs between 4 and32 μg/mL. These P. aeruginosa isolates with knownMICs are mainly used to verify the accuracy of the valuenear the ceftazidime-avibactam breakpoint.All isolates were removed from a − 80 °C ultra-low

temperature freezer and transferred to Columbia bloodagar twice before antimicrobial susceptibility testing.

Antimicrobial susceptibility testingFor the disk diffusion method, ceftazidime-avibactamdisks were obtained from Oxoid (Hampshire, UK). Thecontent of ceftazidime-avibactam in each disk was

30 μg/20 μg. Testing were performed precisely accordingto the recommendations of the CLSI [19].For the Etest gradient diffusion method, ceftazidime-

avibactam Etest strips were obtained from BioMérieux(Marcy l’Etoile, France). The tests were performed in strictaccordance with the manufacturer’s instructions. The cef-tazidime concentration gradient ranged from 0.016 to256 μg/mL with avibactam at a constant concentration of4 μg/mL. When the Etest MIC value was between thestandard value and twice the standard value (0.016, 0.032,0.064, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256),the high standard value was considered as the MIC.The MH agar plates used for both the disk diffusion

method and Etest gradient diffusion method for anti-microbial susceptibility testing were obtained from Oxoid.The BMD was performed strictly following CLSI

guidelines [18]. Ceftazidime and avibactam powder wereobtained from MedChemExpress (Monmouth Junction,NJ, USA). The ceftazidime concentration ranged fromthe standard double dilution of 0.016–256 μg/mL. Theconcentration of avibactam was fixed at 4 μg/mL.Quality controls were evaluated simultaneously in each

batch of experiments. Colony counting was performedto monitor the inoculum density. Escherichia coli ATCC25922, K. pneumoniae ATCC 700603, E. coli ATCC35218, and P. aeruginosa ATCC 27853 were used as ex-perimental quality control isolates. The tests were con-sidered as valid only when the results for all qualitycontrol isolates were within the acceptable range.The MICs and zone diameters of ceftazidime-

avibactam for Enterobacterales and P. aeruginosa wereinterpreted according to the CLSI supplement M10030th edition [18]. Briefly, MICs of ≤8/4 μg/mL or a zonediameter of ≥21 mm indicated that the strain was sus-ceptible, whereas MICs of ≥16/4 μg/mL or a zone diam-eter of ≤20mm indicated that the strain was resistant.Essential agreement (EA) indicated that the difference

between the MIC value measured by Etest and the BMDdid not exceed one two-fold dilution. CA indicated thatinterpretive category results for the Etest method or diskdiffusion method were the same as those for the refer-ence BMD using CLSI breakpoints. VME indicated thatthe strain was susceptible according to the Etest or thedisk diffusion method but resistant according to theBMD. ME indicated that the strain was susceptible bythe BMD but resistant by Etest or the disk diffusionmethod.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s12866-020-01870-z.

Additional file 1: Table S1. Organisms and distribution of carbapenemresistance mechanisms in this study.

Wang et al. BMC Microbiology (2020) 20:187 Page 6 of 7

AbbreviationsBMD: Broth microdilution; MIC: Minimum inhibitory concentration; VME: Verymajor error; CLSI: Clinical & Laboratory Standards Institute; ME: Major error;CA: Categorical agreement; EA: Essential agreement; CRKP: Carbapenem-resistant Klebsiella pneumoniae; CRE: Carbapenem-resistant Enterobacterales;blaKPC: Beta-lactamase gene, Klebsiella pneumoniae carbapenemase;blaNDM: Beta-lactamase gene, New Delhi metallo-β-lactamase; blaIMP: Beta-lactamase gene, imipenemase; ATCC: American Type Culture Collection

AcknowledgementsWe would like to thank BioMérieux for providing Etest strips and Editage(www.editage.cn) for English language editing.

Authors’ contributionsHW conceived and designed the study. QW, FZ, ZW, HC, XW, YZ, and SLperformed experiments described in this study. QW wrote the draft, and HWrevised the manuscript. All authors approved the final version.

FundingHW acknowledges support from the National Natural Science Foundation ofChina (NSFC), Project number: 81625014. The NSFC did not have a role inthe design of the study, in the collection, analysis, or interpretation of thedata, or in writing the manuscript.

Availability of data and materialsAll documents and additional data are available from the correspondingauthor upon reasonable request.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Received: 30 April 2020 Accepted: 22 June 2020

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